Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo - PubMed (original) (raw)

Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo

Ivan B Lobov et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Modulation of Tie2 receptor activity by its angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. It has been proposed that angiopoietins 1 (Ang1) and 2 (Ang2) are pro- and anti-angiogenic owing to their respective agonist and antagonist signaling action through the Tie2 receptor. The function of Ang2 has remained controversial, however, with recent reports suggesting that in some circumstances, it may be pro-angiogenic. We have examined this issue using the transient ocular microvessel network called the pupillary membrane as a unique in vivo model for studying the effects of vascular regulators. We show that in vivo, in the presence of endogenous vascular endothelial growth factor (VEGF)-A, Ang2 promotes a rapid increase in capillary diameter, remodeling of the basal lamina, proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from anti- to pro-angiogenic.

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Figures

Fig 1.

Ang2 promotes cell division in the pupillary membrane. (A) Graph showing the number of mitotic capillary cells in the pupillary membrane after injection of BSA (blue bar), VEGF-A (green bar), or Ang2 (red bar). (B) Experimental timeline and graph describing the increased BrdUrd labeling of pupillary membrane cells 24 h after injection of BSA (blue bars) or Ang2 (red bars). Error bars in A and B are standard errors. Individual and paired BrdUrd-positive cells were counted according to the labeling patterns observed in micrographs (C and D) in which PM cells were exposed to BSA (C) or Ang2 (D). The white arrows indicate pairs of daughter cells and the green arrowheads individual labeled cells. Other labeled nuclei belong to macrophages. Images at ×200.

Fig 2.

Ang2 induces rapid capillary diameter increase and EC shape change. Micrographs of whole-mount control (A) and Ang2-treated (B) PMs showing capillary diameter increase at 24 h (×100). (C) Graph showing the measured increase in capillary diameter of Ang2 (red bar) vs. BSA-treated (blue bar) pupillary membrane capillaries. Error bars are standard errors. (D–G) Higher magnification (×400) paired brightfield and Hoechst 33258-labeled fluorescence micrographs showing PMs exposed to BSA (D), Ang2 (E), Ang2 + anti-Tie2 antibodies (F), and VEGF-A (G). (E) Increased vessel diameter and cell number is apparent; (F) no change is observed; and (G) increased cell number is apparent in the absence of vessel diameter increase. (H–K) Fluorescence micrographs showing anti-VE-cadherin antibody staining (green) for control (H) and Ang2-treated PMs at 4 (I), 12 (J), and 24 h (K) after Ang2 injection. The progressive change in cell shape is apparent from 4 h onward. This is emphasized by the typical cell outlines that are produced in red both on the cell image and projected onto a black area of the micrograph. (H–K) Images at ×630.

Fig 3.

Ang2 induces pericyte-independent EC shape changes and remodeling of the basal lamina. Control (A) and Ang2-treated (B) PM preparations were labeled with Hoechst 33258 for nuclei (blue) and stained with anti-VE-cadherin (green) and antismooth muscle α-actin antibodies (red). This shows that Ang2-induced shape changes occur in capillaries with and without pericytes. Images at ×630. Control (C) and Ang2-treated (D) PM preparations were labeled with Hoechst 33258 for nuclei (blue) and with the HUI77 antibody against a cryptic collagen epitope (red). The higher level of signal present in the dilated capillaries shown in D indicates that the basal lamina is remodeled in response to Ang2. Images at ×400.

Fig 4.

Ang2 stimulates EC migration and sprouting. Micrographs show Ang2-treated PM preparations labeled fluorescently with Hoechst 33258 for nuclei (blue) and anti-VE-cadherin antibody (green). In all panels, cells that can be identified morphologically as macrophages are labeled with a red dot. ECs are identified through junctional immunoreactivity with anti-VE-cadherin antibodies. These panels show typical examples of membranes where ECs have migrated from the confines of the original capillaries. (A) The outline of capillaries is indicated by the white dashed line and groups of cells that have undergone migration by the red dashed lines. These lines are omitted from other panels to afford an unobstructed view. Cells that have migrated form either small groups (A, dashed red lines, and C–E) or complete monolayers between capillaries (B and D). ECs that have migrated often show junctional labeling that is punctate rather than the continuous line found in capillaries (A, B, and D, white arrowheads for examples). Responsive ECs feature cell extensions that label at a low level with anti-VE-cadherin antibodies (C–E, orange arrowheads). In some membranes (F), EC sprouting can be identified where a VE-cadherin junction-positive cell (red arrow) has extended pseudopodia (white arrowheads). (A–E) Images at ×630. (F) Image at 400×.

Fig 5.

Capillary regression is induced by VEGF inhibition and promoted by Ang2. (A) Histogram showing the number of apoptotic capillary cells during the normal regression phase at A9 after BSA injection (blue bar), VEGF injection (green bar), or Ang2 injection (red bar). (B) Histogram showing the number of apoptotic capillary cells during the normal regression phase at A5 after injection of 0.5 μl of the PBS/0.1% BSA vehicle (blue bar), 1 ng of VEGF (green bar), combined 1 ng of VEGF + 20 ng of Flt-Fc (green/yellow bar), 10 ng of Ang2 (red bar), combined 10 ng of Ang2 + 20 ng of Flt-Fc (red/yellow bar), and 20 ng of Flt-Fc (yellow bar). Error bars in A and B are standard errors. (C and D) Paired differential interference contrast (Upper) and Hoechst 33258 fluorescent staining (Lower, false colored green) micrographs showing PMs exposed to BSA (C, control) and Ang2 + Flt-Fc (D). Cells with apoptotic nuclear morphology are indicated (Lower) by the red arrowheads and the pattern of apoptotic cell distribution transferred to the differential interference contrast image. (Insets) A higher magnification of the indicated areas to emphasize apoptotic morphology. Within the Insets, arrows point to apoptotic bodies. Main images at ×400.

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Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo - PubMed (original) (raw)